Microbiology of pollen and bee bread : taxonomy and
enzymology of molds
M. Gilliam, D. B. Prest, B. J. Lorenz
To cite this version:
M. Gilliam, D. B. Prest, B. J. Lorenz. Microbiology of pollen and bee bread : taxonomy and
enzymology of molds. Apidologie, Springer Verlag, 1989, 20 (1), pp.53-68.
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Original article
Microbiology of pollen and bee bread :
taxonomy and enzymology of molds*
M. Gilliam, D. B. Prest
B. J. Lorenz
US Department of Agriculture, Agricultural Research Service, Carl
2000 E. Allen Road, Tucson, AZ 85719, USA
Hayden Bee Research Center,
(received 26-5-1988, accepted 18-8-1988)
Summary — One-hundred and forty-eight molds were isolated from the following samples of
almond, Prunus dulcis, pollen : floral pollen collected by hand; corbicular pollen from pollen traps
placed on colonies of honey bees, Apis mellifera, in the almond orchard; and bee bread stored in
comb cells for one, three, and six weeks. The majority of molds identified were Penicillia (32%),
Mucorales (21%), and Aspergilli (17%). In general, the number of isolates decreased in pollen as it
was collected and stored by the bees. Each type of pollen sample appeared to differ in regard to
mold flora and dominant species. Aureobasidium pullulans, Penicillium corylophilum, Penicillium
crustosum, and Rhizopus nigricans were among the molds that may have been introduced by bees
during collection and storage of pollen. Mucor sp., the dominant mold in floral pollen, was not found
in corbicular pollen and bee bread. Tests for 19 enzymes revealed that most of the molds produced
caprylate esterase-lipase, leucine aminopeptidase, acid phosphatase, phosphoamidase, B-glucosidase, and Macetyl-B-glucosaminidase. Thus, enzymes involved in lipid, protein and carbohydrate
metabolism were produced by pollen molds. Molds could also contribute organic acids, antibiotics
and other metabolites.
pollen
-
bee bread
-
molds
Résumé — Microbiologie du pollen et du pain d’abeilles : taxonomie et enzymologie des moisissures. A l’aide de divers milieux microbiologiques possédant des pH différents, on a isolé 148
moisissures des échantillons suivants de pollen d’amandier, Prunus dulcis : pollen de fleurs récolté
à la main; pollen en pelotes prélevé dans les trappes à pollen posées sur des colonies d’abeilles
(Apis mellifica) dans un verger d’amandiers; et pain d’abeilles stocké dans les cellules des rayons
durant une, 3 et 6 semaines. La majorité des moisissures identifiées sont des Penicillia (32%), des
Mucorales (21 %) et des Aspergillia (17%). C’est le pollen de fleurs qui est le plus riche en isolats,
mais le plus pauvre en espèces. En général le nombre d’isolats diminue dans le pollen quand il est
récolté et stocké par les abeilles. Chaque type d’échantillon pollinique semble différer des autres
par la flore de moisissures et les espèces dominantes. Puisque les moisissures sont identifiées
d’après les besoins de croissance et la caractérisation microscopique et macroscopique des structures morphologiques, les données biochimiques ne proviennent pas des tests taxonomiques. On a
*
Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty
USDA and does not imply its approval to the exclusion of other products or vendors that may
also be suitable.
by the
donc analysé 19 enzymes chez 78 isolats, représentant 28 espèces, par le système API ZYM.
Aucune moisissure ne produit de trypsine, de 8-glucuronidase, ni de a-mannosidase. La plupart
des moisissures produisent de la caprylate lipase-estérase, de la leucine aminopeptidase, de la
phosphatase acide, de la phosphoamidase, de la 8-glucosidase et de la N-acétyle-l3-glucosaminidase. Les moisissures du pollen produisent donc des enzymes impliqués dans le métabolisme des
protéines, des lipides et des glucides.
Ces résultats suggèrent que la flore de moisissures du pollen en pelotes et du pain d’abeilles
peut résulter d’inoculations microbiennes par les abeilles et de modifications chimiques du pollen
dues aux substances ajoutées par les abeilles lors de la régurgitation du contenu du jabot et à la
fermentation microbienne, qui permet à certaines espèces de survivre et à d’autres pas. Même si,
dans nos échantillons, les moisissures étaient plus nombreuses que les espèces de Bacillus et les
levures, le pollen est rarement envahi par elles. Parce qu’elles sont susceptibles de fournir des
enzymes, des acides organiques, des antibiotiques et d’autres métabolites, les moisissures méritent des études plus approfondies.
pollen
-
pain d
abeilles
-
moisissures
Zusammenfassung — Mikrobiologie von Pollen und Bienenbrot : Taxonomie und Enzymologie des Schimmels. Unter Verwendung verschiedener mikrobiologischer Medien mit unterschiedlichem pH-Wert wurden 148 Schimmelpilze von den folgenden Proben von Mandelpollen (Prunus
dulcis) untersucht : Blütenpollen (von Hand gesammelt), Pollenhöschen (aus Pollenfallen an Bienenvölkern [Apis mellifera] im Mandelbaumgarten) und Bienenbrot, das 1, 3 und 6 Wochen in der
Wabe gespeichert war.
Die am häufigsten auftretenden Schimmelpilze waren Penicillia (32%), Mucorales (21 °l) und
Aspergilli (17%). Der Blütenpollen lieferte die meisten Isolate, aber die wenigsten Arten. Im allgemeinen nahm die Anzahl der lsolate im Pollen mit dem Sammeln und Speichern durch die Biene
ab. Jeder Pollentyp schien im Hinblick auf die Schimmelflora und die dominierenden Arten verschieden zu sein.
Da die Schimmelpilze
aufgrund ihrer Wachstumserfordernisse sowie mikrosltopischer wie
makroskopischer Charakterisierung von morphologischen Strukturen identifiziert werden, erhält
man von taxonomischen Untersuchungen keine biochemischen Daten. Daher wurden 78 Isolate,
die 28 Arten repräsentieren, mit dem API ZYM-System auf 19 Enzyme analysiert. Kein Schimmelpilze produzierte Trypsin, β-Glucuronidase oder a-Mannosidase. Die meisten Schimmelpilze produzierten Caprylat-Esterase-Lipase, Leucin-Aminopeptidase, saure Phosphatase, Phosphoamidase,
β-Glucosidase und N-Acetyl-β-Glucosaminidase. Dies bedeutet, daß die untersuchten Pollenschimmel Enzyme des Protein-, Fett- und Kohlehydratstoflwechsels produzieren.
Diese Ergebnisse deuten darauf hin, daß die Schimmelflora im Höselpollen und im Bienenbrot
ein Ergebnis folgender Einflüsse ist : mikrobielle Inokulation und chemische Veränderung des Pollens durch Zugabe von Honigmageninhalt durch die Biene; Drüsensekretion sowie mikrobielle Fermenta6on, die manche Schimmelarten tolerieren, andere nicht. Obwohl Schimmelpilze in unseren
Proben weit zahlreicher waren als Bacillus spp. oder Hefen, wurde der Pollen selten von Schimmel
überwachsen. Die Schimmelpilze sollten als potentielle Spender von Enzymen, organischen Säuren, Antibiotika und anderer Metabolite intensiver untersucht werden.
Pollen
-
Dienenbrot
-
Schimmel
Introduction
Studies have shown for many years that
pollen and bee bread, that is pollen stored
in comb cells of the hive, differ biochemically, and extensive analyses have been
conducted on various floral and bee-collected (corbicular) pollens. The conversion of pollen to bee bread has often been
postulated to be the result of microbial
action, principally a lactic acid fermentation caused by bacteria and yeasts
(Foote, 1957; Haydak, 1958). However,
the chemical and biochemical changes
occurring in pollen as it is collected and
stored by honey bees, Apis mellifera, are
not clearly understood, and relatively little
is known about the microbiology of pollen
and bee bread.
To better understand the nutrition of
honey bees, we studied the chemical, biochemical, and microbiological composition
of pollen from a single plant species before, during, and after storage in comb
cells. Previous papers on the subject by
researchers at the Carl Hayden Bee
Research Center reviewed earlier work
and reported results concerning yeasts
(Gilliam, 1979a); Bacillus ssp. (Gilliam,
1979b); fatty acids, sterols, vitamins, titratable acidity, minerals (Loper et al., 1980);
and protein content, amino acids, selected
enzymes, pH, and 10-hydroxy-!2-decanoic acid (Standifer et al., 1980). In all this
work, the same samples of almond, Prunus dulcis (Prunus communis), pollen
were utilized.
Even though molds are widely known
for their abilities to degrade and to synthesize numerous compounds including the
production of many materials important to
the drug, food and chemical industries,
they have received scant attention in apicultural research concerned with pollen
and bee bread. Early mycological research recognized that certain molds are
common
saprophytes on and inside
honey bees and brood combs, but efforts
were concentrated on dead bees; combs,
particularly from dead colonies; and moldy
pollen (Betts, 1912; Burnside, 1927).
Betts (1912) reported a species of Cladosporium as well as Mucor erectus in corbicular pollen and Beftsia alvei, Eremascus
fertilis, Gymnoascus setosus, Oospora
favorum, and Penicillium
crustaceum in
pollen stored in combs. She noted that
honey appeared to be immune to attacks
of molds. Burnside (1927) stated that
most of the fungi collected by widespread
of honey bees are probably
unable to become established within the
bee or the hive. He found that Penicillia
were the most common molds within the
hive, Aspergilli occurred less frequently,
and species of Mucor did not grow well on
brood combs.
foraging
of provisions
mortality of honey bees are rare
(Batra et aL, 1973). Recently Gilliam and
Vandenberg (1988) reviewed the literature
on fungi pathogenic or detrimental to
honey bees. Only Ascosphaera apis
Fungus-caused spoilage
and
which
causes
chalkbrood
disease
in
honey bees is of economic importance.
The pollen mold, Bettsia alvei, is not a
serious problem since it does not grow
well in cells that are filled with pollen and
finished with a layer of honey on top
(Skou, 1972).
Burri (1947) stated that pollen is germfree in blossoms that have not opened as
well as in opened blossoms if uncontaminated by insect visitation or air currents.
Neither of the two microbiological studies
of pollen and bee bread (Chevtchik, 1950;
Pain and Maugenet, 1966) gave data on
molds, although Chevtchik (1950) mentioned B. alvei as a possible consumer of
lactic acid in bee bread.
(1967) isolated Absidia
Aspergillus flavus, Aspergillus
fumigatus, Aspergillus niger, Aspergillus
terreus, Aspergillus versicolor, Mucor
mucedo, Penicillium clavigerum, PenicilArizan et al.
ramnosa,
lium purpurogenum, Rhizopus nigricans
and Trichothecium roseum from Indian
corn pollen collected by machine. Sainger
et aL (1978) reported that Altemaria
alternata was the most common isolate in
pollen from 3 herbaceous annual plants.
Other molds isolated were Aspergillus
flavus, Aspergillus luchuensis, Aspergillus nidulans, Aspergillus sulphureus, A.
versicolor, Cladosporium oxysporum,
Epicoccum purpurascens, Fusarium
oxy-
sporum, Monilia fructigena, Monilia sitophila, Monilia sp., Rhizopus sp., R. nigricans and Trichoderma viride. Molds isolated from bee bread by Egorova (1971)
were A. flavus, A. versicolor, Mucor
alboalter, Penicillium granulatum, Penicillium solitum and Sporotrichum olivecum.
The present paper reports the results
of the isolation and identification of molds
from almond floral pollen, corbicular pollen, and bee bread stored in comb cells;
analyses of enzymes produced by selected isolates; and comparison of species
isolated with those previously reported
from honey bees in Arizona.
Materials and Methods
Details concerning bee colonies and collections of pollen and bee bread are given by Gilliam (1979a), Loper etal. (1980), and Standifer
et al. (1980). The following samples of almond
pollen were examined : fresh floral pollen collected by hand; corbicular pollen containing
99.8% almond pollen from pollen traps placed
on bee colonies in the almond orchard; and
bee bread stored in cells for 1, 3, and 6 weeks
in newly drawn combs of colonies of bees
maintained in a greenhouse. Each sample was
divided into 4 sub-samples of approximately
0.75 g each. Then each of the 4 sub-samples
was homogenized by hand in 2.5 ml of sterile
0.85% NaCi in a glass tissue grinder. The
homogenates were plated (0.1 ml) in duplicate
on acidified yeast extract-malt extract agar
containing 1% glucose, pH 3.7—3.8 (Miller et
al., 1976); mycological agar with low pH (Difco,
pH 4.8); nutrient agar (Difco) acidified with 0.1
N HCI to pH 5.0; and eugon agar (Difco, pH
7.0). One plate from each sub-sample was
incubated at 25°C and one at 37°C. All were
incubated under aerobic conditions except
eugon agar plates which were placed in 4%
. During a 2-week incubation period, plates
2
C0
were examined periodically for mold growth.
When molds appeared, they were transferred to plates of Czapek solution agar or malt
extract agar (Difco) to allow time for sporulation
and to test for purity. These plates were incubated at 25°C under aerobic conditions. Pure
cultures of isolates were lyophilized for preservation until tests were conducted. Molds were
tested and identified according to Neergaard
(1945), DeVries (1952), Cooke (1959), Ames
(1961), Booth (1961), Morton and Smith (1963),
Ellis (1965), Raper and Fennell (1965), Raper
and Thom (1968), Zycha et al. (1969), Kendrick
and Carmichael (1973), Samson (1974), von
Arx (1975) and McGinnis et al. (1986).
Since molds, in contrast to bacteria and
are identified on the basis of growth
requirements and microscopic and macroscopic characterization of morphological structures,
biochemical data do not result from tests for
identification. Therefore, selected isolates were
tested for 19 enzymes with the API ZYM system (Analytab Products) using the methods of
Bridge and Hawksworth (1984). Suspensions
of some isolates such as Mucorssp. and Alternaria tenuis were sonicated for 1—10 min to
separate fungal spores before inoculation into
the API ZYM strips. Also, malt extract agar
and/or potato dextrose agar (Difco) were used
to prepare inocula of some cultures when
yeasts,
growth appeared
to be less than
optimal
on
Czapek solution agar.
Results
No attempt was made to determine whether spores or mycelial elements were
isolated from pollen and bee bread. However, molds were isolated on all 4 media
used (Table I). Seventy-seven percent of
the isolates were from media incubated at
25°C, and 23% were from media at 37°C
which is near the brood nest temperature
of 34°C (Dunham, 1929). However, the
optimum temperatures for most fungi are
in the range of 20-30°C (Alexopoulos,
1962). Isolations from floral pollen
increased with decreasing pH of the
media. In contrast, the highest percent of
isolations from corbicular pollen from the
pollen trap was on acidified nutrient agar
with a pH of 5.0. Few isolations were
made from floral or corbicular pollen on
eugon agar with a pH of 7.0, but this
situation changed with bee bread. The
percent of isolations
on various media
similar in bee bread samples stored
for one week and for 3 weeks. These
results indicate that a variety of media
with different chemical compositions and
pH values incubated at both 25°C and
37°C aerobically and under C0
2 should
be used for determining mycoflora of pollen and bee bread.
was
One-hundred forty-eight molds were
isolated from pollen and bee bread, of
which 139 were identified (Table II). Overall, the majority of molds identified were
Penicillia (32%), Mucorales (21%), and
Aspergilli (17%). Floral pollen yielded
the highest number of isolates but the
smallest number of species. In contrast,
bee bread stored in comb cells for 3
weeks had the fewest isolates but the
greatest number of different species. In
general, the number of isolates decreased
in pollen as it was collected and stored by
bees.
The most frequent isolate was Mucor
sp., associated exclusively with floral pollen. All 19 isolates appeared similar, but
species identification was not made. They
were characterized by the production of
coenocytic mycelium, some sympodially
branched sporangiophores containing
(chlamydospores) and yellow
globules, globose sporangia with finely
spinose walls, columella with a distinct
collarette, and elliptical smooth sporangiogemmae
spores. The other molds identified from
floral pollen were found in at least one
additional pollen source.
The second most frequently isolated
mold was Penicillium corylophilum. It was
associated with corbicular pollen and all
bee bread sources but not with floral pollen. This was also the case for R. nigricans. Other species which first appeared
in corbicular pollen and were then found
in bee bread were Aureobasidium pullulans, Cladosporium herbarum, Penicillium chrysogenum, and Penicillium crustosum.
Aspergillus niger
isolate and
was a
frequent
found in all types of pollen
samples except bee bread stored for one
week and was isolated most often from
bee bread stored for 6 weeks. Another
frequently encountered mold was Penicillium cyclopium which was most abundant
in bee bread stored for one week and was
found in all sample types except corbicular pollen. Cladosporium cladosporioides
was the only species isolated from all
sample types but was most prevalent in
floral pollen. Molds isolated from more
was
than one source of bee bread but not floral or corbicular pollen were Aspergillus
amstelodami, A. flavus, and Paecilomyces varioti. Alternaria tenuis was found
in floral pollen and bee bread stored for
one week, and Penicillium brevi-compactum was in floral and corbicular pollen but
not bee bread. Other than Chaetomium
elatum which was found in bee bread
stored for 6 weeks, the remaining species
that were identified appeared once in only
one type of pollen source other than floral
pollen. Thus, each type of pollen sample
appeared to differ in regard to mold flora
and dominant species since the predominant mold in almond floral pollen was
Mucor sp.; in corbicular pollen, P. corylophilum and P. crustosum were most common; in bee bread stored in comb cells for
one week, P. cyclopium and P. corylophilum were the most numerous isolates; in
bee bread stored for 3 weeks, there was
no obvious dominant species; and in bee
bread stored for 6 weeks, A.
niger
was
most common.
unidentified molds in Table 11
non-viable after lyophilization. We
were also unable to assign the unidentified Ascomycete to genus. It produced
ostiolate dark ascocarps with unbranched
terminal hairs. The asci were cylindrical
and contained 4 ascospores. Ascospores
Eight
were
ellipsoidal to ovoid, smooth, nonapiculate, and yellow-brown to brown.
Since it was difficult to determine by light
microscopy whether the ascospores were
single or double-pored, scanning electron
microscopy was used to reveal that the
majority were single-pored, although
double-pored ascospores were also pres-
were
ent.
To determine enzymatic activity of the
molds from pollen and bee bread,
attempts were made to test at least one
strain of each species and more strains of
the
species frequently isolated. However,
Cladosporium sphaerospermum did not
survive lyophilization after identification
and could not be tested. Thus, 78 isolates
representing 28 species were each analyzed for 19 enzymes. A total of 113 complete tests were conducted owing to replication of tests, use of additional media for
preparing the inocula of strains which did
not grow well on Czapek solution agar,
duplicate tests which were incubated as
long as 24 h, and tests comparing enzymology of spore and mycelial inocula of
selected strains. Aspergillus amstelodami, Chaetomidium pilosum, Chaetomium
elatum, Thielavia sepedonium, Xylohypha bantiana, and the unidentified Ascomycete failed to grow well enough on
Czapek solution agar to yield sufficient
inocula for API ZYM tests and were therefore grown on potato dextrose agar and
malt extract agar and then tested for
enzymes. Other selected species were
also grown
on
various media, and the
enzymology was compared to inocula
from Czapek solution agar. Results of
tests on inocula of the same strain preon different media gave the same
results except that the concentrations of
one or two of the enzymes produced were
in a few cases slightly higher when the
growth medium was potato dextrose agar
compared to malt extract agar. These differences could be related to improved
growth of the molds and/or to the media
composition. Bridge and Hawksworth
(1984) also found some minor variations
with different media. We also noted a few
similar minor variations when the incubation time was extended for 24 h for strains
of R. nigricans. Mycelial inocula produced
the same enzymes as spore suspensions,
although in smaller quantities.
pared
Results of enzymatic activities based
identities of the isolates regardless of
the pollen source are shown in Tables
on
III-VI. All 40 Penicillia tested produced
leucine aminopeptidase, acid phosphatase, phosphoamidase, and f3-glucosidase;
none produced cystine aminopeptidase,
trypsin, a-galactosidase, f3-glucuronidase,
or a-mannosidase (Table 111). Most also
produced alkaline phosphatase, caprylate
esterase-lipase, and N-acetyl-B-glucosaminidase. Enzymes produced by Penicillia in the highest concentrations (> 20
nmol) were acid phosphatase by P. corylophilum, N acetyl-f3-glucosaminidase by
P. cyclopium, and f3-glucosidase by P.
crustosum.
All Aspergilli
tested produced acid
phosphatase, phosphoamidase, B-glucosidase, and N acetyl-f3-glucosaminidase;
none produced myristate lipase, trypsin,
chymotrypsin, f3-glucuronidase, a-mannosidase, or a-fucosidase (Table IV). Most
also produced alkaline phosphatase, butyrate esterase, caprylate esterase—lipase,
and leucine aminopeptidase. One of the
A. flavus strains tested was var. columnaris and gave the same reactions as strains
identified as A. flavus. Enzymes produced
in the highest concentrations (> 20 nmol)
by Aspergilli were N-acetyl-B-glucosaminidase and fi-glucosidase by A. niger, alkaline phosphatase by A. flavus, and
alkaline and acid phosphatases and Bglucosidase by A. versicolor.
All Murocales tested produced leucine
aminopeptidase, and most produced
acid phosphatase and phosphoamidase
(Table V). Otherwise these molds produced few enzymes. The only enzymes
produced in high concentrations (> 20
nanomoles) were phosphoamidase by all
strains of R. nigricans tested, acid phosphatase by 3 of the 4 R. nigricans strains,
and leucine aminopeptidase by Mucor
racemosus.
Of the other molds tested, most produced acid phosphatase and f3-glucosidase (Table VI). Alkaline phosphastase,
butyrate esterase, caprylate esterase—
lipase, leucine aminopeptidase, and
phosphoamidase were produced by
52—67% of them. Enzymes produced in
the highest concentrations (> 20 nmol) by
various molds
follows : alkaline
Aur. pullulans from one-
are as
phosphatase by
week bee bread; caprylate esterase—lip—
ase by C. herbarum from corbicular pollen
and by C. cladosporioides; leucine aminopeptidase by Aur. pullulans and Peyronelia sp.; acid phosphatase by Alt. tenuis,
Arthrinium phaeospermum, Aur. pullulans, C. cladosporioides, C. herbarum,
Peyronelia sp., Scytalidium sp., and X.
bantiana; phosphoamidase by X. bantiana; a-galactosidase by Aur. pullulans from
one-week bee bread and by C. cladosporioides from corbicular pollen; B-glucosi-
dase by Alt. tenuis and T. sepedonium
and by C. cladosporioides from 6-week
bee bread; and a-fucosidase by C. cladosporioides from floral and corbicular
pollen.
Enzymology of the
presented in Table Vil
78 molds tested is
on the basis of the
of the isolates. No molds
pollen sources
produced trypsin, f3-glucuronidase, or amannosidase. Few produced myristate
lipase, and only one produced chymotrypsin; these isolates
were
from corbicular
pollen. Valine aminopeptidase and cystine
aminopeptidase were not produced by
isolates from 3-week bee bread, nor was
the latter enzyme produced by molds from
corbicular or 6-week bee bread. Few
molds from other pollen sources produced
these two peptidases. Of the glycos
i
dases, a-glucosidase and a-fucosidast.
were not associated with molds from
either 3-week or 6-week bee bread and
produced by few isolates from other
pollen sources. Both a- and B-galactosidases were produced by a higher percent
of molds tested from floral pollen than
from other pollen sources. No molds from
corbicular pollen produced B-galactosi-
were
dase.
most molds from all pollen
produced caprylate esterase-
However,
sources
lipase, leucine aminopeptidase, acid
phosphatase, phosphoamidase, B-glucosidase, and N acetyl-f3-glucosamidase. A
high percent of the isolates (> 50%) from
all sources gave positive reactions for
alkaline phosphatase. This was also the
case with butyrate esterase except for
those molds from one-week bee bread.
Therefore, pollen molds produced
enzymes involved in protein, lipid, and
carbohydrate metabolism.
lum, P. crustosum, and R. nigricans.
Conversely, Mucor sp., the dominant mold
in floral pollen, was eliminated in corbicular pollen and bee bread. Thus, as with
yeasts (Gilliam, 1979a) and Bacillus ssp.
(Gilliam, 1979b), the mold flora of corbicular pollen and bee bread may be the
result of microbial inoculations by bees
and chemical changes in pollen resulting
from additions by bees from regurgitation
of honey sac contents and secretions of
glands as well as microbial fermentation
which allow some species but not others
to survive. Even though molds were more
than yeasts or Bacillus ssp. in
samples, pollen is rarely overgrown by
molds. Potential microbial spoilage of pollen provisions may be controlled by antibiotic substances produced by the normal
microflora, bees, pollen, and/or honey.
numerous
our
Klungness and Peng (1983) examined
pollen and bee bread with
electron
microscopy and found
scanning
no visible evidence of digestion or damage to pollen grain walls. They concluded
that microorganisms associated with bee
bread do not cause destruction of pollen
intine or the cytoplasm, that substances
bees add to pollen during collection and
storage function as a preservative, and
that the regurgitation added to pollen
probably allows growth of some microorganisms and inhibits the growth of others.
They observed that the few fungal spores
that germinated produced hyphae less
than 10 pm in length.
If microorganisms are responsible for
fermentation and the accompanying
chemical changes of pollen stored in
comb cells by honey bees, the molds may
be a component of the required microbial
complement. They could contribute antibiotics, organic acids and enzymes, products for which they are utilized industrially. These compounds may limit the growth
of deleterious microorganisms and provi-
corbicular
Discussion
Seventy percent of the molds identified
pollen and bee bread were
and Penicillia. We
Mucorales,
Aspergilli,
have previously isolated from apiarian
from almond
in Arizona most of the species of
and Penicillia found in pollen
and bee bread as well as Aur. pullulans,
C. claedosporioides, Mucorales, and
Peyronelia sp. (Gilliam and Prest, 1972,
1977, 1987; Gilliam et al., 1974, 1977,
sources
Aspergilli
1988). Frequent isolates in the present
study which are new records of molds
from apiarian sources in Arizona are
Alt. tenuis, Penicillium crustosum, and
R. nigricans.
Molds which were not present in floral
pollen but were frequent isolates from corbicular pollen and bee bread may have
been introduced by the bees during collection and storage. The most obvious
examples are Aur. pullulans, P. corylophi-
de enzymes for utilization of nutrients.
Indeed, we have found Aspergilli, Mucorales, Penicillia, and other molds in bee
bread and guts of worker bees which inhibit the
growth of the chalkbrood fungus
(Gilliam et aL, 1988).
Enzymology of our isolates revealed
that the major phosphatase was acid
phosphatase, although alkaline phosphatase
was
produced by
most
isolates
except the Mucorales. Molds apparently
are not able to participate in the initial
breakdown of long-chain fatty acids as
evidenced by the lack of myristate lipase.
However, they did produce butyrate
and
es-
caprylate esterase—lipase
which act on shorter chain fatty acids.
Leucine aminopeptidase was the major
aminopeptidase. Molds did not produce
trypsin or chymotrypsin. Phosphoamidase was produced by most isolates tested.
Results for glycosidases revealed that
Mucorales were quite unreactive. Howrase
ever, most other molds
produced B-gluco-
sidase which hydrolyzes carbohydrates
such as cellobiose, salicin, amygdalin,
and gentibiose. N Acetyl-f3-glucosaminidase was also produced by most molds.
This enzyme is involved in hydrolysis of
chitin. f3-Galactosidase which hydrolyzes
lactose and a-glucosidase which hydrolyzes sucrose, maltose, trehalose, and
melizitose were not produced by most of
the molds.
In summary, molds as normal microflora in pollen and bee bread have received little attention. However, our results
indicate that because they represented
38% of the total number of microorganisms we isolated
unpublished
data), produced a variety of enzymes, and
are well known for production of secondary metabolites such as antibiotics, phenolic compounds, terpenes, steroids, and
polysaccharides as well as enzymes, they
(Gilliam,
merit more intensive study. Even if mold
spores that germinate in corbicular pollen
and bee bread produce short hyphae
(Klungness and Peng, 1983), our results
with selected isolates of various genera
confirmed those with Penicillia by Bridge
and Hawksworth (1984) that mycelia inocula produce th,
same
enzymes,
in
reduced
as spore
amounts,
although
suspensions of the same strain. With this
publication and those on yeasts (Gilliam,
1979a) and Bacillus ssp. (Gilliam, 1979b),
we have now reported 77% of the total
isolates from our almond pollen and bee
bread samples. The remaining microorganisms will be described in a future publication.
Mycological Papers, No. 83, Commonwealth
Mycological Institute, Kew, England
Bridge P.D. & Hawksworth D.L. (1984) The API
ZYM enzyme testing system as an aid to the
rapid identification of Penicillium isolates.
Microbiol. Sci. 1, 232-234
Burnside C.E. (1927) Saprophytic fungi associated with the honey bee. Mic;1.Acad. Sci. 8,
59-86
Burri R.
(1947) Die Beziehungen der Bakterien
Lebenszyklus der Honigbiene. Schweiz.
Bienen-Ztg. 70, 273-276
Chevtchik V. (1950) Mikrobiologie pylov6ho
kvaseni. Publ. Fac. Sci. Univ. Masaryk 323,
zum
103-130
Cooke W.B. (1959) An ecological life
Aureobasidium pullulans (deBary)
history of
Arnaud.
Mycopathologia 12, 1-45
DeVries G.A. (1952) Contribution to the Knowlege of the Genus Cladosporium Link ex Fr.
Centraalbureau voor Schimmelcultures, Baarn,
The Netherlands
Acknowledgments
We thank Dr. L.N. Standifer of the Carl Hayden
Bee Research Center for the pollen samples,
Dr. James T Sinski of the University of Arizona
for providing scanning electron microscopy and
consultation on the unidentified Ascomycete,
and Drs. E.W. Herbert Jr., D.R. Jimenez, and
J.D. Vandenberg for reviewing the manuscript.
Dunham W.E. (1929) The relation of external
temperature on the hive temperature during the
summer.
J. Econ. Entomol. 22, 798-801
Egorova A.I. (1971) Preservative microflora in
stored pollen. Veterinariya 8, 40-41
Ellis M.B. (1965) Dematiaceous Hyphomycetes. Vi. Mycological Papers, No. 103, Commonwealth Mycological Institute, Kew, England
Foote H.L. (1957) Possible use of microorganisms in synthetic bee bread production. Am.
Bee J. 97, 476-478
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